Aristolochic Acid Content Research Articles

Preparation of Anti-Aristolochic Acid I Monoclonal Antibody and Development of Chemiluminescent Immunoassay and Carbon Dot-Based Fluoroimmunoassay for Sensitive Detection of Aristolochic Acid I.

Aristolochic acid (AA) toxicity has been shown in humans regarding carcinogenesis, nephrotoxicity, and mutagenicity. Monitoring the AA content in drug homologous and healthy foods is necessary for the health of humans. In this study, a monoclonal antibody (mAb) with high sensitivity for aristolochic acid I (AA-I) was prepared. Based on the obtained mAb, a chemiluminescent immunoassay (CLEIA) against AA-I was developed, which showed the 50% decrease in the RLUmax (IC50) value of 1.8 ng/mL and the limit of detection (LOD) of 0.4 ng/mL. Carbon dots with red emission at 620 nm, namely rCDs, were synthesized and employed in conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) to improve the assay sensitivity of a fluoroimmunoassay (FIA). Oxidized 3,3′′,5,5′′-tetramethylbenzidine dihydrochloride (oxTMB) can quench the emission of the rCDs through the inner-filter effect; therefore, the fluorescence intensity of rCDs can be regulated by the concentration of mAb. As a result, the assay sensitivity of FIA was improved by five-fold compared to CLEIA. A good relationship between the results of the proposed assays and the standard ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-QQQ-MS/MS) of real samples indicated good accuracy and practicability of CLEIA and FIA.

Chemotaxonomy of the ethnic antidote Aristolochia indica for aristolochic acid content: Implications of anti-phospholipase activity and genotoxicity study

Ethnopharmacological relevanceAristolochia indica L. (Aristolochiaceae) is a common medicinal plant described in many traditional medicine as well as in Ayurveda used against snakebites. Besides, the plant has also been reported traditionally against fever, rheumatic arthritis, madness, liver ailments, dyspepsia, oedema, leishmaniasis, leprosy, dysmenorrhoea, sexual diseases etc. The plant is known to contain its major bioactive constituent aristolochic acid (AA) known for its anti-snake venom, abortifacient, antimicrobial and antioxidant properties. Materials and methodsThis present work describes a validated, fast and reproducible high performance thin layer chromatography (HPTLC) method to estimate AA from the roots of 20 chemotypes of A. indica procured from 20 diverse geographical locations from the state of West Bengal, India. Further, an evidence-based approach was adopted to investigate the reported anti-venom activity of the aqueous extracts of the A. indica roots by assessing its phospholipase A2 (PLA2) inhibitory properties since PLA2 is a major component of many snake-venoms. Finally, the cytotoxicity and genotoxicity of the aqueous root extract of the Purulia (AI 1) chemotype were assessed at various concentrations using Allium cepa root meristematic cells. ResultsThe highest amount of AA (7643.67 μg/g) was determined in the roots of A. indica chemotype collected from Purulia district followed by the chemotypes collected from Murshidabad, Jalpaiguri and Birbhum districts (7398.34, 7345.09 and 6809.97 μg/g respectively). This study not only determines AA in the plants to select pharmacologically elite chemotypes of A. indica, but it also identifies high AA producing A. indica for further domestication and propagation of the plants for pharmacological and industrial applications. The method was validated via analyzing inter-day and intra-day precision, repeatability, reproducibility, instrumental precision, limit of detection (LOD) and limit of quantification (LOQ) and specificity. Chemotypes with high AA content exhibited superior anti-PLA2 activity by selectively inhibiting human-group PLA2. Moreover, A. indica root extract significantly inhibited mitosis in Allium cepa root tips as a potent clastogen. ConclusionsThe present quick, reproducible and validated HPTLC method provides an easy tool to determine AA in natural A. indica plant populations as well as in food and dietary supplements, a potential antivenin at one hand and a possible cause of aristolochic acid nephropathy (AAN) at another. Besides, the cytotoxic and mitotoxic properties of the root extracts should be used with caution especially for oral administration.

Abstract A20: Dietary ingestion of aristolochic acid: Mechanisms of exposure

Abstract Aristolochic acids (AA) are human carcinogens and nephrotoxins found in Aristolochia plants, including herbs used in traditional medicines throughout the world. Aristolochic acids can also be ingested as an environmental contaminant in food, which forms the basis of Balkan endemic nephropathy and associated upper tract urothelial cancer. Aristolactam DNA-adducts, formed after metabolism of AA, are promutagenic and are resistant to repair and readily detected in human tissue sample DNA decades after exposure. Thus, AL-DNA adducts are excellent biomarkers of exposure to this carcinogen. The mutational signature of AA is distinct for an environmental carcinogen and is recognized as Signature 22 in the COSMIC mutational signature database. Molecular epidemiology using the presence of adducts and/or the presence of Signature 22 has implicated AA in the etiology of several cancers, including upper-tract urothelial cancer, bladder cancer, renal cell clear cell carcinoma, hepatocellular carcinoma, and cholangiocarcinomas. In regions of the world reliant on traditional medicines, including much of Asia, the AA contribution to incidence of these cancers is significant. In the Balkan countries of Europe, the route of exposure is thought to occur via contamination of wheat harvests with Aristolochia plant material from weed growth. In endemic villages farming families traditionally have ingested primarily wheat flour milled from their own harvests. An alternative hypothesis is that decaying Aristolochia plants release the chemically stable aristolochic acids into the soil, and these are then taken up and concentrated by food plants. To distinguish between these hypotheses, we have obtained 25 flour samples from individual farms along the Croatia/Bosnia and Herzegovina border. To identify AA-contaminated flour, aristolochic acids were extracted from aliquots of the flour, reduced to aristolactams, and quantitated with an LC-ESI-MS/MS method. Several flour samples were indeed contaminated with AA in the range of 10–100 parts per billion. Secondly, we also extracted DNA from each flour sample. The direct contamination hypothesis predicts the presence of Aristolochia DNA in contaminated flour samples; the indirect contamination via decay hypothesis does not. Using widely accepted plant “barcode” PCR protocols, we amplified a portion of the rbcLa gene with generic primers that amplify the gene from all species. Within this PCR amplicon each plant species has a constellation of specific base-pairs. Thus, the contribution of all plant species to the flour sample can be determined by highly parallel sequencing of the amplicon. We will present the results of this next-gen sequencing to determine the contribution of Aristolochia to Balkan flour samples and correlation with aristolochic acid content. Citation Format: Viktoriya Sidorenko, Robert Rieger, Bojan Jelakovic, Thomas Rosenquist. Dietary ingestion of aristolochic acid: Mechanisms of exposure [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A20.

Cloning of AhWRKY33 from Asarum heterotropoides and its genetic transformation in Arabidopsis

The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.

Does group feeding by toxic prey confer a defensive benefit? Aristolochic acid content, group size and survival of first‐instar pipevine swallowtail (Battus philenor L.) larvae

1. Aggregative feeding of larvae is widespread in the Lepidoptera, and many hypotheses have been proposed to explain the adaptive significance of this trait. Group feeding occurs disproportionately more in species with aposematic, chemically defended larvae compared with species with cryptic, non‐chemically defended larvae, consistent with the hypothesis that group feeding provides an enhanced aposematic signal to natural enemies. Most species characterised as having chemically defended larvae are cryptic during the first instar, when they are most highly aggregated and most vulnerable to predation.2. The benefits of group feeding in terms of decreased predation were explored for first‐instar larvae of the pipevine swallowtail, Battus philenor, a species that sequesters aristolochic acids from its Aristolochia host plant and exhibits aposematism in later instars and as adults. We found that groups of larvae with experimentally enhanced aristolochic acid content had significantly lower survivorship due to predation both in the field and in laboratory experiments compared with groups of larvae without enhanced chemical defence.3. A laboratory experiment found that aristolochic acid does not deter the generalist predator Hippodamia convergens.4. No evidence was found that was consistent with the hypothesis that group feeding and increased sequestered chemical defence interact to decrease rates of larval mortality in non‐aposematic, first‐instar larvae. Future research on chemical defence, aposematism, and aggregative feeding should continue to appreciate that particular chemical defences and feeding behaviours are not universally effective against all natural enemies.

The effects of qualitative and quantitative variation of aristolochic acids on preference and performance of a generalist herbivore

AbstractNearly all plants possess chemicals that are inferred to play a role in anti‐herbivore defense or resistance. The effects of various chemical defenses can vary among herbivores. Often, plant defensive compounds are examined in broad, inclusive categories, with an emphasis on total quantity, which might ignore qualitative variation in activity. Aristolochic acids are alkaloids characteristic of plants of the genus Aristolochia (Aristolochiaceae). Although aristolochic acids have been documented as effective herbivore deterrents, it remains unknown whether different kinds of aristolochic acid vary in their efficacy as defense against herbivores. We manipulated the aristolochic acid content of artificial diet to examine the effects of four aristolochic acids on larval preference and performance of the generalist herbivore Spodoptera exigua Hübner (Lepidoptera: Noctuidae). Using choice tests, we observed that the four aristolochic acids tested varied in their deterrent effectiveness, with AA‐I having the strongest effect and AA‐II having the weakest effect. No‐choice tests were used to examine larval performance. The effect on performance varied among the aristolochic acids tested. Higher concentrations of aristolochic acid were generally associated with reduced larval developmental rate and larger size at pupation. These results indicate that various forms of aristolochic acid can vary in their effect on herbivores and that simple aggregate measures of total concentration might not reflect the chemical defensive phenotype of the plant.

Association Between Aristolochic Acid and CKD: A Cross-sectional Survey in China

Long-term intake of herbs containing aristolochic acid (AA) has been reported to be associated with increased risk of chronic kidney disease (CKD), whereas population-based studies are limited. Cross-sectional study. A national representative sample of 47,204 adults in China. Self-reported long-term use of medications containing AA. CKD was defined as estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m(2) and/or the presence of albuminuria, defined as albumin-creatinine ratio >30 mg/g. 467 participants reported long-term AA intake, with a prevalence adjusting for a synthesized weight of 1.5% (95% CI, 1.2%-1.7%). After adjusting for age, sex, and other potential confounders, long-term AA intake was associated with eGFR <60 mL/min/1.73 m(2) and albuminuria, with ORs of 1.83 (95% CI, 1.22-2.74) and 1.39 (95% CI, 1.03-1.87), respectively. Further adjustment for intake of nonsteroidal anti-inflammatory drugs did not change ORs substantially. A positive association between accumulated time of AA intake and kidney disease also was observed, with fully adjusted ORs of 1.07 (95% CI, 1.03-1.12) per 6-month longer intake for eGFR <60 mL/min/1.73 m(2) and 1.04 (95% CI, 1.01-1.08) per 6-month longer intake for albuminuria. Self-reported intake of herbs containing AA; the AA content of the preparations by weight was unknown; single measurement of indicators of kidney damage. Long-term intake of medications containing AA is prevalent in China and is associated with the presence of CKD.

Characterization of the aqueous extract of the root of Aristolochia indica: Evaluation of its traditional use as an antidote for snake bites

Ethnopharmacological relevanceThe aqueous extract of the roots of Aristolochia indica is used as a decoction for the ailment of a number of diseases including snake bite treatment. Though the alcoholic extract of the different parts of the plant are well studied, information on the aqueous extract is limited. We have estimated aristolochic acid, different enzymes, enzyme inhibitors and anti-snake venom potency of its root extract. Materials and methodsReverse phase–HPLC was used to quantify aristolochic acid. Zymography, DQ-gelatin assay and atomic force microscopy were done to demonstrate gelatinase and collagenase activities of the extract. SDS-PAGE followed by MS/MS analysis revealed the identity of major protein components. Toxicity of the extract was estimated on animal model. Interaction of the extract with Russell's viper venom components was followed by Rayleigh scattering and enzyme assay. ResultsThe aristolochic acid content of the root extract is 3.08±1.88×10−3mg/ml. The extract possesses strong gelatinolytic, collagenase, peroxidase and nuclease activities together with l-amino acid oxidase and protease inhibitory potencies. Partial proteomic studies indicated presence of starch branching enzymes as major protein constituent of the extract. The extract did not show any acute and sub-chronic toxicity in animals at lower doses, but high dose causes liver and kidney damage. The extract elongated duration of survival of animals after application of Russell's viper venom. ConclusionsConsidering the low aristolochic acid content of the extract, its consumption for a short time at moderate dose does not appear to cause serious toxicity. Strong inhibition of l-amino acid oxidase may give partial relief from snake bite after topical application of the extract.

Family matters: effect of host plant variation in chemical and mechanical defenses on a sequestering specialist herbivore

Insect herbivores contend with various plant traits that are presumed to function as feeding deterrents. Paradoxically, some specialist insect herbivores might benefit from some of these plant traits, for example by sequestering plant chemical defenses that herbivores then use as their own defense against natural enemies. Larvae of the butterfly species Battus philenor (L.) (Papilionidae) sequester toxic alkaloids (aristolochic acids) from their Aristolochia host plants, rendering larvae and adults unpalatable to a broad range of predators. We studied the importance of two putative defensive traits in Aristolochia erecta: leaf toughness and aristolochic acid content, and we examined the effect of intra- and interplant chemical variation on the chemical phenotype of B. philenor larvae. It has been proposed that genetic variation for sequestration ability is "invisible to natural selection" because intra- and interindividual variation in host-plant chemistry will largely eliminate a role for herbivore genetic variation in determining an herbivore's chemical phenotype. We found substantial intra- and interplant variation in leaf toughness and in the aristolochic acid chemistry in A. erecta. Based on field observations and laboratory experiments, we showed that first-instar larvae preferentially fed on less tough, younger leaves and avoided tougher, older leaves, and we found no evidence that aristolochic acid content influenced first-instar larval foraging. We found that the majority of variation in the amount of aristolochic acid sequestered by larvae was explained by larval family, not by host-plant aristolochic acid content. Heritable variation for sequestration is the predominant determinant of larval, and likely adult, chemical phenotype. This study shows that for these highly specialized herbivores that sequester chemical defenses, traits that offer mechanical resistance, such as leaf toughness, might be more important determinants of early-instar larval foraging behavior and development compared to plant chemical defenses.

Verbot von Asarum – eine fragwürdige Sicherheitsphilosophie

Xi xin – radix et rhizoma asari – is a classical drug in Chinese medicine which is vitally important to various of applications that make it difficult to replace. After it became public in the 1990s that aristolochic acid (AA)-I and –II can cause nephropathia and urothelic carcinoma, aristolochic acid content of asarum species received quite some attention. The AA-I as well as AA-II contents documented in medical literature are actually low, in some cases even undetectable. Asarum crispulatum however, which is not officinal, is characterized by an extremely high AA content. Due to this, in 2010 the use of asarum species was banned in Germany and Switzerland. The Federal Institute for Drugs and Medicinal Devices (BfArM) concedes that during a 2-year use, a 50 % life time tumor risk is only tolerable in 1 out of 1,000,000 persons. Compared to conventional drugs as well as to every day life risks, the safety threshold in this case seems unusually low. By applying more realistic calculations similar to, for instance, the casualty risk for the entire population posed by the use of nuclear energy in Germany per annum, health risks could be controlled by limiting the duration of use, as well as enforcing strict quality control measures on xi xin drugs. A total ban of asarum species, however, seems inappropriate.

Simple and rapid analysis of aristolochic acid contained in crude drugs and Kampo formulations with solid-phase extraction and HPLC photodiode-array detection

Simple and rapid analysis of aristolochic acid (AA) in crude drugs and Kampo extracts using a solid-phase extraction method and HPLC-PDA analysis was investigated. Extraction of AA from samples was accomplished by adding methanol containing 1% ammonia. The addition of ammonia ionized the AA of acidic substances so that they adhered to an acrylamide copolymer of a strong anion exchange resin (Sep-Pak QMA) coupled to diol silica easily. Furthermore, a mixture of acetonitrile-water-phosphoric acid (75:25:2, v/v) was effective in isolating AA from its carrier. Since almost all interfering peaks originating from contaminants in crude drugs and Kampo extract formulations could be removed, a satisfactory HPLC chromatogram of AA was obtained. A good result was also obtained when Aristolochiaceae and crude drugs containing AA were tested. Particularly in the case of the medicinal parts of Asarum, several interfering peaks and a ghost peak detected near the AA peak were eliminated. The AA contents of two Kampo extract formulations, tokishigyakukagoshuyushokyoto and ryutanshakanto, were calculated by HPLC analysis. The AA content (the sum of AA-I and AA-II) was 1.25-6.13 mg per daily dose. From an additional recovery experiment for Kampo formulations, high recovery rates of AA were obtained. Neither LC/MS nor special instrumentation was necessary. Our results suggest that this simple, quick, and sensitive analytical method to detect AA in crude drugs and Kampo extract formulations would be valuable in safety inspections of AA in crude drugs and their products.

Aristolochic acids affect the feeding behaviour and development of Battus polydamas archidamas larvae (Lepidoptera: Papilionidae: Troidini)

The feeding behaviour of specialist butterflies may be affected by the mechanical and chemical characteristics of the tissues of their host-plants. Larvae of the butterfly, Battus polydamas archidamas feed only on Aristolochia chilensis, which contains aristolochic acids. We studied the oviposition pattern of adults and the foraging of larvae of B. polydamas archidamas over time in relation to variations in hardness of the substrate and concentration of aristolochic acids in different plant tissues. We further tested the effect of two artificial diets containing different concentrations of aristolochic acids on larval performance. B. polydamas archidamas oviposited mostly on young leaves and the larvae fed on this tissue until the second instar. Third instar larvae fed also on mature leaves and fourth and higher instars fed also on stems. Young leaves are softer and contain higher concentrations of aristolochic acids than mature leaves, and stems are both harder and contain a high concentration of aristolochic acids. Larvae reared on artificial diets containing a high concentration of aristolochic acids suffered less mortality and were heavier than those reared on a diet with a lower concentration of aristolochic acids, which suggests they are phagostimulatory. A strategy of host use regulated by aristolochic acid content and tissue hardness is discussed.

ANTAGONISTIC, STAGE‐SPECIFIC SELECTION ON DEFENSIVE CHEMICAL SEQUESTRATION IN A TOXIC BUTTERFLY

Larvae of the pipevine swallowtail (Battus philenor) sequester toxic alkaloids called aristolochic acids from their Aristolochia host plants, rendering both larvae and adults chemically defended against most predators. Using a chemically controlled artificial diet, we observed substantial among-family variation in sequestration ability and larval developmental rate in a population occurring in central Texas. Early instar larvae from families that sequester greater amounts of aristolochic acid showed increased survivorship in a field experiment in which cohorts from each family were exposed to natural predators, whereas among-family variation in growth rate did not predict survivorship. Conversely, the aristolochic acid content of adult butterflies was negatively correlated with adult fat content, a fitness correlate. Sequestration ability positively affects the probability of larval survivorship, but at the cost of adult fat content. The costs and benefits of aristolochic acid sequestration vary during the course of the butterfly's development, and these antagonistic selection pressures may explain why variation in sequestration ability persists in wild populations.

Aristolochic Acids Detected in Some Raw Chinese Medicinal Herbs and Manufactured Herbal Products – A Consequence of Inappropriate Nomenclature and Imprecise Labelling?

Background. Certain frequently used Chinese herbal medicines commonly used for weight control, may contain toxic Aristolochia species, which have been associated with severe nephropathy and urothelial cancer in humans and animals. The toxic entities in Aristolochia species are aristolochic acid-I (AA-I) and aristolochic acid-II (AA-II). There is a lack of systematic information about the aristolochic acid content of Aristolochia species and related genera, including those in Chinese materia medica that are used in the treatment of overweight individuals. Objectives. To determine the content of AA-I and AA-II of commonly used Chinese herbal medicines (raw herbs and manufactured products) including species of Aristolochia and related genera. Methods. Twenty-one raw herbs and seven manufactured herbal products were purchased from herbal wholesalers and traditional Chinese medicinal herb retailers in Melbourne, Australia in September 2003, after the supply of known aristolochic acid-containing herbs and products had been banned in Australia. Six additional raw herbs were sourced from a herbal teaching museum. These were purchased in 2001, before the prohibition. The contents of aristolochic acids of each was determined by high pressure liquid chromatography (HPLC), and confirmed by liquid chromatography–mass spectrometry (LC–MS). Results. Of the samples tested, four of the raw herbs purchased before the ban and two manufactured products purchased after the ban, were found to contain aristolochic acids (16–1002 ppm). Conclusions. Several Chinese raw herbs and some commercially available manufactured herbal products contain aristolochic acids. The confusion in Chinese nomenclature for related raw herbs, and imprecise labelling of manufactured products may contribute to the inadvertent use of toxic herbal species in Chinese medicine practice. Additional measures are needed to ensure the safety of consumers of Chinese herbal medicines.

Phenological Variation in Chemical Defense of the Pipevine Swallowtail, Battus philenor

Larvae of the pipevine swallowtail, Battus philenor, feed on plants in the genus Aristolochia, which contains aristolochic acids, toxic alkaloids unique to the Aristolochiaceae. Pipevine swallowtails sequester these compounds and, as a consequence, are chemically defended against many natural enemies. In California, the primary aristolochic acid present in the butterfly is aristolochic acid I. Newly eclosed adult females possess greater amounts of these sequestered toxins compared to males. However, over the course of the flight season, the aristolochic acid content of females in the population declines, whereas male aristolochic acid content remains relatively constant. Transference of sequestered aristolochic acids to eggs by females might explain the decline of these sequestered chemical defenses observed over time. We found no evidence that males transfer aristolochic acids to females via the spermatophore. The possibility that females at the end of the flight season may be automimics of males is discussed. Temporal variation in the aristolochic acid defenses exhibited by this pipevine swallowtail population is both age- and sex-dependent.

A Model Without a Mimic: Aristolochic Acids from the California Pipevine Swallowtail, Battus philenor hirsuta, and Its Host Plant, Aristolochia californica

The pipevine swallowtail, Battus philenor, feeds exclusively on plants in the genus Aristolochia, many of which are known to contain the toxic alkaloids collectively known as aristolochic acids. Pipevine swallowtails sequester these compounds and use them for their own defense against predators. Numerous palatable butterflies are involved in Batesian mimicry complexes with B. philenor over its range. The California subspecies of the pipevine swallowtail, B. philenor hirsuta, has no mimics. Analysis of the butterfly and its only host plant, Aristolochia californica, indicate that both contain aristolochic acid. Aristolochic acid (I) and (II) are the primary aristolochic acids found in A. californica. The highest concentration of aristolochic acids was found in the flowers, which bloom before B. philenor emerges. Concentrations of aristolochic acids decreased in the leaves but not in stem tissue over the course of the season. Butterflies contained primarily aristolochic acid (I). Aristolochic acid content of individuals from Arizona, which are involved in mimicry complexes, did not differ from California populations. Thus, lack of California mimics cannot be attributed to low aristolochic acid content of the model.